1. Field of the Invention
The invention relates to methods for determining nucleic acid sequences or peptide sequences that bind biologically active macromolecules. More particularly, the invention relates to the use of hybridization to oligonucleotide arrays to determine nucleic acid sequences or binding to peptide arrays to determine amino acid sequences involved in peptide binding.
2. Summary of the Related Art
The determination of the nucleotide sequence of nucleic acids and the amino acid sequence of peptides and proteins is central to many critical analyses in molecular biology. Such sequence determination allows investigation of mutations that are responsible for inherited diseases and neoplastic conditions. These determinations potentially allow comparison of genetic alleles responsible for variation in human inherited traits. Finally, such determination allow identification of the precise nature of molecular interactions necessary for a plethora of critical biological or biochemical functions. These promises have led to proposals for ambitious projects involving macromolecule sequence determination, such as the proposed sequencing of the entire human genome. Consequently, procedures for sequencing nucleic acids and peptides have developed rapidly and become well known in the art. Nevertheless, existing procedures for sequence determination are sufficiently time consuming to limit the speed at which ambitious sequencing projects can proceed. This state of affairs has led to proposals for newer, nonconventional sequencing procedures that are more rapid than existing techniques. One promising idea is to utilize hybridization to determine nucleotide sequences.
Proposals for the direct sequencing of DNA by hybridization with arrays of oligonucleotides are known in the art. Drmanac et al., Genomics 4: 114 (1989) proposes hybridization array-mediated DNA sequencing by binding target DNA to a dot blot membrane, followed by probing with an array of oligonucleotides. Khrapko et al., FEBS Letters 256: 118 (1989) proposes hybridization array-mediated DNA sequencing by binding the oligonucleotide array to a support membrane, followed by probing with target DNA.
Synthesis of arrays of bound oligonucleotides or peptides is also known in the art. Houghton, in the Multiple Peptide System product brochure describes the T-bag method, in which an array of beads is physically sorted after each interaction. This method becomes unwieldy for the preparation of large arrays of oligonucleotides. Geysen et al., J. Immunol. Methods 102: 259 (1987) discloses the pin method for the preparation of peptide arrays. The density of arrays that may be produced by this method is limited, and the dipping procedure employed in the method is cumbersome in practice. Southern, Genome Mapping and Sequencing Conference, May 1991, Cold Spring Harbor, N.Y., disclosed a scheme for oligonucleotide array synthesis in which selected areas on a glass plate are physically masked and the desired chemical reaction is carried out on the unmasked portion of the plate. In this method it is necessary to remove the old mask and apply a new one after each interaction. Fodor et al., Science 251: 767 (1991) describes a method for synthesizing very dense 50 micron arrays of peptides (and potentially oligonucleotides) using mask-directed photochemical deprotection of synthetic intermediates. This method is limited by the slow rate of photochemical deprotection and by the susceptibility to side reactions (e.g., thymidine dimer formation) in oligonucleotide synthesis. Khrapko et al., FEBS Letters 256: 118 (1989) suggests simplified synthesis and immobilization of multiple oligonucleotides by direct synthesis on a two dimensional support, using a printer-like device capable of sampling each of the four nucleotides into given dots on the matrix. However, no particulars about how to make or use such a device are provided.
Some methods for permanently attaching oligonucleotides to glass plates in a manner suitable for oligonucleotide synthesis are known in the art. Southern, Chem. Abst. 113: 152979r (1990) describes a stable phosphate ester linkage for permanent attachment of oligonucleotides to a glass surface. Mandenius et al., Anal. Biochem. 157: 283 (1986) teaches that the hydroxyalkyl group resembles the 5′-hydroxyl of oligonucleotides and provides a stable anchor on which to initiate solid phase synthesis.
A variety of procedures for synthesis of oligonucleotides are, of course, known in the art. Matteucci et al., Nuc. Acids Res. 14: 5399 (1986) discloses an H-phosphonate protocol that uses excess pivaloyl chloride/pyridine for activation, thereby providing a self-capping coupling step.
Special apparatus is required to deliver minute quantities of materials precisely for the preparation of oligonucleotide arrays. Some information about piezoelectric pumps is known in the photographic art. Kyser et al., J. Applied Photographic Engineering 7: 73 (1981) teaches that energizing a piezoelectric element deforms the cavity much like a one-sided bellows. Parameters for oligonucleotide hybridization also are known in the art. Drmanac et al., DNA and Cell Biology 2: 527 (1990) teaches that maximum selectivity in hybrid formation occurs near the Tm1/2 of an 8-mer, or about 25° C., and that selectivity decreases at higher temperatures. McGraw et al., BioTechniques 8: 674 (1990) teaches that the bond energy of a GC pair is nearly twice that of an AT pair in low ionic strength SSC buffers. Wood et al., Proc. Natl. Acad. Sci. USA 82: 1585 (1985) teaches that 3 M trimethylammonium chloride stabilizes the AT base pair sufficiently to make the Tm1/2 of a hybrid independent of base composition, and dependent only on the number of mismatches.
In summary, the related art contains numerous ideas and information related to the synthesis of arrays of oligonucleotides or peptides for the determination of nucleotide sequences or the amino acid sequences of specific binding peptides. However, existing or suggested methods are limited, and do not conveniently and reliably produce the very large, high density arrays necessary for effective large-scale sequencing. There is, therefore, a need for new methods for preparing large high density arrays of oligonucleotides or peptides for sequencing based upon oligonucleotide hybridization or peptide binding Ideally, such methods should utilize relatively simple machinery to produce large, dense arrays of solid phase bound oligonucleotides or peptides in a reproducible and rapid manner.